A novel thermostable GH5 β-xylosidase from Thermogemmatispora sp. T81

Abstract

A glycoside hydrolase family 5 (GH5) subfamily 22 gene, designated T81Xyl5_22A, was identified in the genome of the aerobic thermophilic bacterium, Thermogemmatispora sp. T81 (locus A4R35_07040). The gene was cloned and heterologously expressed in Escherichia coli and the gene product characterized biochemically. The recombinant enzyme had an optimal catalytic activity at pH5.0 and 65 °C, and was active against beechwood xylan and rye arabinoxylan. It yielded only xylose molecules as products of beechwood xylan hydrolysis, indicating that it is a GH5 family β-d-xylosidase. Using 4-nitrophenyl β-d-xylopyranoside (pNPX) as a substrate, the K_M, Vmax, k_cat and k_cat/K_M kinetic parameters were determined as 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s^-1 and 156.8 mM^-1 s^-1, respectively. Small-angle X-ray scattering (SAXS) data enabled reconstruction of the enzyme's low-resolution molecular envelope and revealed that it formed dimers in solution. As far as we are aware, this is the first description of a thermostable bacterial GH5 family β-d-xylosidase.

Keywords: Enzyme kinetics; Enzyme structure; Heterologous gene expression; Thermogemmatispora sp. T81; Thermostability.